Patterns of aeroallergen sensitization in asthma patients identified by latent class analysis: A cross‐sectional study in China

Abstract Background This cross‐sectional study aimed to identify latent sensitization profiles of asthma patients in mainland China, unveiling the association between regional differences and sensitization patterns. Methods 1056 asthma participants from 10 medical centers divided into eastern and western cohorts were clustered into four individual sensitization patterns, respectively, by using an unsupervised statistical modeling method, latent class analysis (LCA), based on the levels of 12 aeroallergens specific IgE reactivities. Moreover, differences in clinical characteristics and environmental exposures were compared in different sensitization patterns. Results Four distinct sensitization patterns in the two cohorts were defined as follows, respectively. Eastern cohort: Class 1: “High weed pollen and house dust mites (HDMs) sensitization” (8.87%), Class 2: “HDMs dominated sensitization” (38.38%), Class 3: “High HDMs and animal dander sensitization” (6.95%), Class 4: “Low/no aeroallergen sensitization” (45.80%). Western cohort: Class 1: “High weed pollen sensitization” (26.14%), Class 2: “High multi‐pollen sensitization” (15.02%), Class 3: “HDMs‐dominated sensitization” (10.33%), Class 4: “Low/no aeroallergen sensitization” (48.51%). Of note, the significant statistical difference in age, asthma control test score (ACT) and comorbidities were observed within or between different sensitization patterns. Exposure factors in different sensitization patterns were pointed out. Conclusions Asthmatic patients with distinct sensitization patterns were clustered and identified through the LCA method, disclosing the relationship between sensitization profiles of multiple aeroallergens and geographical differences, providing novel insights and potential strategies for atopic disease monitoring, management and prevention in clinical practice.


| INTRODUCTION
Globally, about 300 million patients were affected by asthma currently, 1 which was one of the most common inflammatory disorders of the airways worldwide. Roughly estimated, the prevalence of asthma in people over 20 years was 4.26% in China, reaching 45.7 million in total, posing a tremendous social and economic burden. 2 As a primary causative factor, exposure and sensitization to aeroallergens are responsible for most of the onset and persistence of asthma to a large extent. 3 Alternations have been observed in patterns of aeroallergens sensitization in China in the last decade, demonstrating that geographical differences affect asthmatic patients sensitized. 4 Besides, previous studies focused more on the northern or southern coastal areas and school-age children, 5 while little is known about the differences in aeroallergens sensitization patterns in the western and southwestern regions in mainland China and lack large-scale epidemiology research in different age periods. Furthermore, quite a part of asthma patients is ignorant of themselves having co-sensitization to multiple allergens, thus making it more difficult to control and manage in clinical practice. 6

| Patients and study design
This cross-sectional study was conducted in 10 medical centers in China between September 2021 and October 2022, including 5 eastern regions (Jiangsu, Shandong, Guangdong, Henan, and Hebei) and 5 western regions (Gansu, Sichuan, Ningxia, Inner Mongolia, and Shaanxi). Patients can be diagnosed as asthma if he/she meets the following symptoms and meets any of the objective tests for airflow limitation, except wheezing, shortness of breath, chest tightness and cough caused by other diseases: (a) recurrent wheezing, shortness of breath, with or without chest tightness or cough, frequent at night and in the morning; (b) sporadic or diffuse wheezing sounds could be heard in both lungs; (c) objective inspection of variable airflow limitation: bronchodilation test was positive, positive bronchial excitation test, peak expiratory flow (PEF) was >10%, or the weekly variation rate of PEF was >20%. Patients who at least sensitized to one of the allergens in Skin prick test (SPT) (Grade 1-4) or sIgE detection (>0.35 kU/L) will be diagnosed as allergic asthma (AA). After excluding the missing data, 1056 asthmatic patients aged 0-86 years were recruited, according to the guidelines of the Global Initiative for Asthma (GINA) 8 and Chinese. 9 After signing informed consent, patients underwent an SPT with 16 aeroallergens and collected 5 mL venous blood for serum-sIgE detection of 12 aeroallergens. Furthermore, patients and/or their legal guardians filled in a questionnaire. The study design was presented in Figure 1 and created with BioRender.com.

| Standardized questionnaire
The questionnaire consisted of basic information, clinical history,  five questions about asthma control as listed below. 10 (i) How often has asthma influenced your daily activities (work/study/rest) in the past four weeks? (ii) How many times have you had difficulty breathing (anhelation, shortness of breath or poor breathing) in the past four weeks? (iii) How many times have you woken up at night or earlier than usual in the morning due to asthma symptoms (wheezing, coughing, dyspnea, chest tightness or pain), in the past four weeks?
(iv) How many times have you used emergency medicine (such as salbutamol) in the past four weeks? (v) How did you assess your asthma control over the past four weeks? According to GINA criteria, the ACT score was classified into three different groups: (i) ACT score<20, uncontrolled; (ii) 20 = ACT score<25, partially controlled; and (iii) ACT score≥25, controlled. All completed questionnaires were verified and double-checked by two well-trained investigators. Phragmites communis). Histamine and normal saline serve as positive and negative controls respectively. All performances followed the standard operation procedure. After 15 min, the wheal reaction was measured as the mean of the maximum diameter and the length of the perpendicular line through its middle. Any allergen showing the size of a wheal ≥3 mm than the negative control should be considered a positive reaction. The result was presented as skin index (SI = mean size of allergen wheal/mean size of histamine wheal). SI accounts for 25%, 50%, 100% and 200% of the histamine-induced wheal area was defined as Grade 1, 2, 3 and 4 respectively. Grades 1-4 were considered as positive skin reactions, whereas Grade 0 was suggested as a negative reaction. All patients enrolled in the study had discontinued antiallergic drugs for at least 14 days before the SPT.

| Detection of the level of sera-specific IgE (sIgE)
Asthma patients were offered blood draw for the detection of 12 aeroallergen IgE, including 6 indoor allergens, D1 (Dermatophagoides

| Characteristics of study participants
In this multicenter study, 417 and 639 asthmatic patients were recruited from the eastern and western regions of China, respectively. The median age of the patients was 20.89 years 461 (43.66%) and 595 (56.34%) participants were female and male respectively.
More specific details were shown in Table 1 and Additional File 1   Table S3.

| Sensitization rates and distributions of 16 representative aeroallergens on SPT
Remarkably, a high sensitization rate to house dust mites (HDMs) of 50.0% was noted in patients in the eastern regions, followed by    Figure 2B). The difference in sensitization rates of different aeroallergens between the two cohorts was statistically significant (***,

| Classification and characteristics of sensitization patterns in different cohorts
In the eastern cohort, 417 participants were clustered into four distinct sensitization categories using the LCA model.   Figure 4A). Additionally, as shown in the Figure 4B-E,  Figure 5A).
In the western cohort, results revealed that the patients of Class probably later than that in the eastern populations ( Figure 5B).

| Differences in ACT score and comorbidities among different sensitization patterns
In the eastern cohort, ACT results demonstrated that asthma patients' scores mainly ranged from 20 to 25, except for the Class 4 population (33.51%, p < 0.05). Concerning the Class 4 population, the non-atopic asthma patients' phenotype may account for the low ACT score, showing a relatively low sensitization (<5.0%) to these 12 aeroallergens ( Figure 6A).
ZHANG ET AL.   While in the western cohort population, more complicated sensitized profiles were discovered, demonstrating a sensitization to weed, tree and grass pollens or HDMs. Compared with patients with similar sensitization patterns in the western cohort, asthmatic patients of Class 1 in the eastern cohort manifested a somewhat higher F I G U R E 7 Risk factors were revealed by multiple logistic regression analysis. In the eastern cohort, compared with Class 4 (reference category), females in Class 1 presented as a protective factor. While in Class 2, natural labor, without Allergic rhinitis (AR) and not using air conditioners are protective factors, but pollen exposure could be a risk factor. In Class 3 populations, living in rural areas or not using mattresses or without furry animal exposure are protective factors, while tobacco smoke exposure is a risk factor ( Figure 7A). In the western cohort, compared with the Class 4 (reference category), living in the suburbs, without AR and furry animal exposure are protective factors in Class 1, whereas do not use air conditioners is a risk factor. In Class 2 populations, not using air conditioners is a risk factor, while without AR, tobacco smoke and pollen exposure, not using the mattress, and living in a high domestic storey (≥9 floors) are protective factors. On the contrary, living in a high domestic storey (≥9 floors) is a risk factor in Class 3 populations. While living in the countryside, without furry animal exposure, not using air conditioners and mattresses are protective factors ( Figure  7B). ZHANG ET AL.  Despite the limitation of the cross-sectional study, it is noted that participants enrolled in our study were of substantial representativeness in general asthmatic patients in China. If possible, additional prospective research will probably be better to determine and verify whether these latent classes conform with corresponding asthma patients clinically. Therefore, further studies need to be explored to reveal the underlying mechanism and association of atopic respiratory diseases, providing novel viewpoints and possible strategies for precision diagnosis and prevention in the future.

| CONCLUSIONS
In summary, based on the levels of sIgE reactivity, our findings indicated that asthma patients with sensitization to multiple respiratory allergens could be clustered and summarized into four distinct sensitization profiles through the LCA statistical modeling method.
Hence, focusing on the western and eastern areas, this multicenter study successfully recognized and unveiled the differences between sensitization patterns, geographical differences and clinical characteristics in asthmatic patients.

AUTHOR CONTRIBUTIONS
Baoqing